Anti-HIV dual specificity antibodies and methods of HIV treatment

ABSTRACT

Dual variable domain immunoglobulins (DVD Igs) are provided capable of tetravalent binding to bispecific sites of the human immunodeficiency virus (HIV). The DVD Igs may be asymmetric and may have more variable domains on either the light chain or the heavy chain of the Igs. The DVD Igs may have specificity for gp41 and gp120. Therapies are provided using DVD Igs to neutralize HIV viral loads.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the 35 U.S.C. § 371 national stage application of PCT Application No. PCT/US2014/047787, filed Jul. 23, 2014, which claims benefit of U.S. Provisional Application 61/858,149 filed Jul. 25, 2013, both of which are incorporated herein by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

The instant application contains a sequence listing which has been submitted with the instant application via EFS-Web. The sequence listing file is named 222271030_ST25.txt and is herein incorporated by reference. The support for the sequences can be found throughout the specification.

FIELD

The present invention relates generally to anti-HIV antibodies and antibody-like molecules and, in particular though non-limiting embodiments, to double-variable domain antibodies and methods of treating an HIV infection.

BACKGROUND

The lentivirus human immunodeficiency virus (HIV) causes Acquired Immunodeficiency Syndrome (AIDS), a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive. HIV/AIDS is a global pandemic, with most recent World Health Organization studies estimating nearly 34 million people are infected with HIV. This figure includes over 3 million children under the age of 15. Currently, no cure for HIV exists.

The HIV virus infects a large number of different cells in the body, including various cell types of the immune system, but its infection of CD4 T-lymphocytes largely underlies HIV pathogenesis. HIV-infection leads to reduced CD4 T-lymphocytes, further leading to progressive loss of cell-mediated immunity and an increased susceptibility to opportunistic infections.

The HIV virus consists of a viral envelope enclosing a capsid, which itself encloses the viral genome. The HIV envelope protein (Env) consists of precursor gp160 of the transmembrane domain gp41 (e.g., SEQ ID NO 1, which is one of many sequences for gp41) and the external domain gp120 (e.g. SEQ ID NO 2, which is one of many sequences for gp120), which are involved in virus-cell attachment. Mechanistically, gp120 attaches to the CD4 molecule present on T-lymphocytes, a series of conformational changes occur with gp120 and gp41, and gp41 mediates the fusion of the viral and cellular membranes and insertion of viral core and the genomic material into the target cell, resulting in host cell infection.

Conventional neutralizing antibodies generally consist of two identical heavy chains and two identical light chains, each with a single variable domain (V_(H) or V_(L)) at the N-termini of the molecule. More recently, neutralizing antibodies have been adapted to include a second variable region connected via a linker (L) sequence at the N-termini of the variable domains of a conventional molecule and are generally referred to as a dual variable domain immunoglobulins (DVD-Igs). DVD-Igs are immunoglobulin-derived molecules that contain two unique variable domains (V domains) linked to a constant region with the capability of tetravalent, bispecific binding, while retaining affinity and specificity of each of the parental antibodies. For example, DVD-Igs have been constructed that can bind both IL1a and IL1b, or IL-12 and IL-18. DVD-Igs have been proven effective in vitro and in vivo, and retain pharmacokinetic properties of the parental antibodies.

The idea of targeting two separate antigenic sites with a single antibody has also been directed against HIV. The most common approach has been to construct dual domain antibodies using an anti-gp120 V-region fused to CD4. When the inter-domain linker length was optimized, enhanced neutralization by these CD4-anti-gp120 immunoadhesins was obtained. Bi-specific antibodies with one V-domain against gp41 and one against gp120 have been produced; however the antibodies do not neutralize the virus as well as embodiments of the present invention. The failure to make effective neutralizing antibodies is due in part to the enormous sequence diversity of HIV-1, and the relative inaccessibility of conserved domains of the HIV virus.

Accordingly, there is need for novel antibodies and antibody like molecules and methods of neutralizing and eradicating HIV.

SUMMARY

In an exemplary embodiment of the present invention, an antibody is provided, including: at least one variable domain with binding affinity to HIV gp120 and at least one variable domain with binding affinity to HIV gp41. At least one of a heavy chain and a light chain of the antibody has a variable domain with binding affinity to HIV gp120 linked by a linker to a variable domain with binding affinity to HIV gp41. In certain embodiments, only one of the heavy chain and the light chain may have two variable domains. In certain embodiments, both the heavy chain and light chain may have two variable domains.

The linker may be one of a helical linker and a flexible linker. The linker may be one of SEQ ID NO:9 and SEQ ID NO:10. The antibody may include both chains of full-length 7B2 antibody. The heavy chain may have one variable domain with binding affinity to HIV gp41 and the light chain may have a first variable domain with binding affinity to HIV gp120 linked by the linker to a second variable domain with binding affinity to HIV gp41. The light chain may have one variable domain with binding affinity to gp41 and the heavy chain may have a first variable domain with binding affinity to HIV gp120 linked by the linker to a second variable domain with binding affinity to HIV gp41. Both the light chain and the heavy chain may have a first variable domain with binding affinity to HIV gp120 linked by the linker to a second variable domain with binding affinity to HIV gp41.

The heavy chain of the antibody may be one of SEQ ID NO 3, SEQ ID NO 4, and SEQ ID NO 8. The light chain of the antibody may be one of SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7. The light chain is not SEQ ID NO 7 when the heavy chain is SEQ ID NO 8. The at least one variable domain with binding affinity to HIV gp120 may include domains 1 and 2 of CD4.

In an exemplary embodiment of the present invention, a method of treating an HIV infection is provided, including: administering to a patient infected with HIV at least one of an antibody and a genetic construct capable of producing the antibody in the patient, said antibody having: at least one domain with binding affinity to HIV gp120; and at least one domain with binding affinity to HIV gp41. At least one of a heavy chain and a light chain of the antibody has a domain with binding affinity to HIV gp120 linked by a linker to a domain with binding affinity to HIV gp41. The antibody may have been incorporated into an immunoconjugate having at least one of a toxin and a cytotoxic agent.

In an exemplary embodiment of the present invention, a method of neutralizing HIV virus is provided, including: administering at least one of an antibody and a genetic construct capable of producing the antibody, said antibody having: at least one domain with binding affinity to HIV 120; and at least one domain with binding affinity to HIV gp41. At least one of a heavy chain and a light chain of the antibody has a domain with binding affinity to HIV gp120 linked by a linker to a domain with binding affinity to HIV gp41. The least one of an antibody and a genetic construct capable of producing the antibody may be administered to a subject infected with HIV. The least one of an antibody and a genetic construct capable of producing the antibody may be administered to a subject at risk of exposure to HIV.

DESCRIPTION OF DRAWINGS

FIG. 1 is a set of three graphs showing binding qualities of constructs and parental antibodies to antigens, according to an exemplary embodiment of the present invention.

FIG. 2 is a set of two graphs showing indirect immunofluorescence and flow cytometry analysis of binding qualities to persistently infected H9/NL4-3 cells, according to an exemplary embodiment of the present invention.

FIG. 3 is a set of two graphs showing indirect immonoconjugate assays of anti-gp120 and anti-gp41/gp120 constructs, according to an exemplary embodiment of the present invention.

FIG. 4 is a set of four graphs showing assay results for neutralization of viral isolates, according to an exemplary embodiment of the present invention.

FIG. 5 is a set of three graphs showing assay results for neutralization of viral isolates, according to an exemplary embodiment of the present invention.

FIG. 6 is a graph showing assay results for antibody-dependent cellular viral inhibition, according to an exemplary embodiment of the present invention.

FIG. 7 is a graph showing assay results for antibody-dependent phagocytosis, according to an exemplary embodiment of the present invention.

DETAILED DESCRIPTION

The present invention relates generally to bispecific antibodies, asymmetric antibodies, antibody-like molecules and methods of treating HIV infections. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by any examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Throughout this disclosure, the term “antibody” may indicate a classical antibody, but also an antibody-like molecule, a protein, a fragment thereof, or any combination of these. “Antibody” may include proteins, fragments, glycoproteins (e.g., CD4), or portions thereof attached or linked to antibodies and/or variable domains of antibodies. Sequences encoding CD4 (domains 1 and 2) are referred to as variable domains in this application. These terms are intended to be illustrative in nature, and certainly not limiting.

Any antibody or antibody fragment of the present invention, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modification or particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property.

Embodiments of the present invention may be administered to a subject in a pharmaceutical composition, which may be any acceptable carrier. Effective dosages and schedules for administering embodiments of the present invention may be determined empirically. Embodiments may be administered to neutralize, treat, prevent or eradicate HIV infection. Embodiments of the present invention may be used in gene therapy techniques for HIV. In certain embodiments, genetic constructs capable of inducing production of antibodies of the present invention may be administered to a patient in need thereof.

Embodiments of the present invention provide an antibody having at least one variable domain with binding affinity to HIV gp120 and at least one variable domain with binding affinity to HIV gp41. At least one of a heavy chain and a light chain of the antibody may have a variable domain with binding affinity to HIV gp120 linked by a linker to a variable domain with binding affinity to HIV gp41.

Embodiments of the present invention include synthetic genes, which encode novel antibody constructs of the present invention. Embodiments of the present invention include monoclonal antibodies to HIV having at least one dual variable domain chain with the dual domains linked by the linker. Embodiments of the present invention may be derived from monoclonal antibody 7B2.

Embodiments of the present invention provide methods of HIV treatment and neutralization. HIV virus may be neutralized and/or eliminated with novel constructs of the present invention. Novel constructs of the present invention may be administered for neutralization of HIV loads and/or may include a cytotoxic agent to trigger cytotoxic activities in infected cells. Embodiments of the present invention provide therapeutic treatments for HIV infections using novel DVD-Ig constructs. Embodiments of the present invention provide methods of preventing HIV infection by administering novel antibody constructs. The novel constructs of the present invention may be administered as part of a pharmaceutical composition or as genetic constructs to be made into antibodies by the patient's own cells.

An embodiment of the present invention includes an antibody having a heavy chain of SEQ ID NO 3 and a light chain of SEQ ID NO 5—construct #2816. An embodiment of the present invention includes an antibody having a heavy chain of SEQ ID NO 4 and a light chain of SEQ ID NO 6—construct #2817. An embodiment of the present invention includes an antibody having a heavy chain of SEQ ID NO 8 and a light chain of SEQ ID NO 5—construct #2858. An embodiment of the present invention includes an antibody having a heavy chain of SEQ ID NO 3 and a light chain of SEQ ID NO 7—construct #2859. An embodiment of the present invention includes an antibody having a heavy chain of SEQ ID NO 8 and a light chain of SEQ ID NO 6—construct #2860. An embodiment of the present invention includes an antibody having a heavy chain of SEQ ID NO 4 and a light chain of SEQ ID NO 7—construct #2861.

It is understood that upon binding of gp120 to CD4, the HIV Env protein on the surface of the virion undergoes a conformational change that exposes several key epitopes on both gp120 and gp41 that are vulnerable to attack by our immune systems. Substantial work has been published on the potent neutralization activity of CD4 binding site (CD4bs) antibodies like VRC01 and b12. This class of antibodies binds to the CD4 binding site on gp120 to block interaction. In addition to blocking the ability of the virus to recognize its cognate receptors, the conformation changes expose areas of gp41 necessary for membrane fusion and cell entry.

It has been known that addition of soluble CD4 (sCD4) enhances immunoconjugate activity by changing the conformation of the Env protein. Recently more CD4-inducible (CD4i) antibodies have been characterized; however, effective neutralization requires the presence of CD4. This led to the problem of how to deliver both a soluble form of CD4 and the monoclonal antibody simultaneously, which was solved by fusing the relevant portion of CD4 to a CD4i-antibody through a variety of linkers. Several CD4i reagents, such as 17b, E51, and m9, have been fused to sCD4 for enhanced activity. A range of CD4-i reagents have been produced from large, bispecific antibodies constructs to smaller single-chain immunoadhesins. While the smaller scFv forms may result in higher tissue penetration and epitope access, they are naturally less avid and limited in their ability to stimulate the immune system through Fc interactions and antibody dependent cell cytotoxicity (ADCC). In the case of CD4-7B2 constructs, the CD4 subunits are not only advantageous for increasing neutralization but enhance exposure of the precise epitope 7B2 requires.

Contemporary fusion antibodies were included in studies alongside a panel of CD4-7B2 DVD-Igs embodiments of the present invention. This allowed for a comparison of the effects of CD4 fusion versus mixing CD4i-antibodies with CD4-IgG. It would be much more efficient to supply a single engineered form of CD4-7B2, especially if it neutralizes an extremely broad range of HIV isolates. In embodiments of the present invention, CD4-7B2 IgGs were engineered to function as both an immunoconjugate and a neutralizing antibody. Results show that fusion of CD4 to a single chain of 7B2 through a linker, such as a flexible linker or a helical linker, creates an optimal configuration for binding the Env subunits, neutralizing the infectivity of the virus, and killing cells already infected and producing the virus.

In addition to neutralization, immunoconjugates (IC) may be beneficial in reducing infectivity. HAART has contributed to a substantial decrease in viral loads, yet there is no vaccine or cure available. A potential approach would be use of an IC to eliminate the latent reservoir through an “activate and purge” strategy advocated by inducing reactivation of integrated viral genomes and subsequent killing of infected cells. An IC is a chimeric protein that combines the targeting specificity of an antibody with the cellular effects of a toxin. Correctly chosen parental antibodies can be incorporated into ICs which bind viral proteins on the surface of infected cells where they are subsequently phagocytized, intracellularly processed, and then activated to kill the infected cell. Certain toxic molecules can induce apoptosis over necrosis which helps reduce spread of virus to neighboring healthy cells. In this protocol, an HIV-activating agent would be administered first, and then treatment with ICs would deliver apoptosis-inducing drugs into infected cells. Early attempts have proved the concept that ICs can effectively kill infected cells in vitro using a CD4bs antibody and a bacterial exotoxin. In example embodiments of the present invention, ricin A chain (RAC) was utilized due to its cytotoxic attributes and relative stability over time. Extensive studies have shown how both native ricin and conjugated ricin are trafficked and activated to kill cells in vitro and in vivo. The new CD4-7B2 constructs of the present invention represent potential therapeutics with an increased breadth of neutralization and the unparalleled cell-targeted killing ability of ICs.

In an embodiment of the invention, the variable domains of the bispecific antibody disclosed herein comprise domains from antibodies that may bind the CD4 binding site, variable loops, and/or glycans of gp120, and the variable domains from the anti-gp41 monoclonal antibody 7B2, or other gp41 antibodies. The CD4-binding site of gp120 may be targeted with either CD4-itself (for example, soluble CD4) or an antibody or fragment thereof specific for this binding site. Such antibody or fragment thereof may be monoclonal in nature. In embodiments of the present invention, the variable domains may be linked by a linker (also known as an inter-V chain linker). Notably, unique construction of the inter-V chain linker may comprise synthetic peptides with either unordered or helical conformations.

Unlike conventional double-domain antibodies, which are symmetric assemblies of two identical heavy chains and two identical light chains, each containing identical variable regions, embodiments of the present invention may comprise asymmetric double variable domain antibodies, wherein the variable regions are not identical. For example, in one embodiment, a heavy chain may contain a V_(H1)-L-V_(H2), and the light chain variable region may consist of V_(L2) only. It should be noted that any combination thereof may be used to make the asymmetric double-variable domain antibodies, such as antibodies that may bind gp120 and gp41. In embodiments of the present invention, symmetric or asymmetric heavy and light chains may be used.

It is generally known that tetrameric, symmetrical CD4-IgG neutralizes better than other CD4-IgG constructs, suggesting that symmetrical CD4-anti-gp41 antibodies may be effective neutralization antibodies. However, the neutralization capabilities of the symmetrical CD4-anti-gp41 antibodies were poor when experimentally tested, suggesting that CD4-anti-gp41 antibodies or antibody-like molecules may be poor candidates for HIV-neutralization antibodies. Surprisingly, when asymmetric CD4-anti-gp41 antibodies were constructed and tested in TZM-b1 cells against a panel of tier 1 and tier 2 clade B and C viruses, such antibodies had astonishingly high neutralization capacity. These surprising results suggest that the asymmetric structure of these antibodies may provide increased neutralization capacity.

An advantage of the disclosed invention is that the antibodies work well as neutralizing antibodies. This is a unique advantage to these antibodies, as other constructs were not as effective as neutralizing antibodies. For example, preliminary tests performed using 7B2-CD4 hybrid IgG molecules in TZM-b1 cells against a panel of tier 1 and tier 2 clade B and C viruses demonstrate that these constructs may be highly effective neutralizing antibodies. The 7B2 antibody served as a reference antibody in these tests, and the CH01-31 antibody served as a positive control. In particular, the results from this experiment demonstrate that CD4-anti-gp41 hybrid proteins disclosed herein are potent neutralizers.

Embodiments of the invention may be employed to treat or prevent HIV. In one such embodiment, the antibodies may be used as immunoconjugates, conjugated to a second molecule. For example, the second molecule may be a toxin, a label, a radioisotope, a drug, or a chemical compound.

In other embodiments, the antibodies disclosed herein may be used as neutralizing antibodies, passively administered or given via gene therapies. Supporting these approaches, preliminary data suggests that certain constructs may be highly effective neutralizing antibodies.

Generally, embodiments of the present invention comprise double variable domain (DVD) antibodies that may bind in a tetravalent fashion to antigens of Env, such as gp120 and gp41. Antibodies to gp41 and/or gp120 of Env may provide important neutralization components necessary for an effective HIV/AIDS therapeutic, as Env is the only HIV protein displayed fully intact on the surface of HIV-infected cells. Embodiments of the present invention may incorporate monoclonal antibody 7B2, which binds to the external loop of gp41. In certain embodiments of the present invention, asymmetrical DVD antibodies are provided.

Embodiments of the present invention provide immunoglobulins (Igs) engineered to improve HIV neutralization and/or delivery of cytotoxic agent to infected cells. Example embodiments include CD4-Igs containing domains 1 and 2 of human CD4 attached to either heavy, light, or both chains of full-length human 7B2 antibody, an IgG1/κ anti-gp41 antibody. Embodiments may be engineered using helical linkers or other flexible sequences. For example, a helix-creating sequence (SEQ ID NO 9) or a combination of helical and flexible sequences (SEQ ID NO 10) may be incorporated as linkers. Embodiments may be created by expressing H and L chains in different plasmids and co-transfecting the H and L chains into 293F cells, creating CD4/7B2 chimeric Igs with various CD4-antibody conformations and linker usage. A description of example embodiments of the present invention is set forth in Table 1, where construct nos. 2816, 2817, and 2858 to 2861 represent novel embodiments of the present invention.

TABLE 1 Description of Parental Abs and Chimeric Constructs Name H Chain L Chain Linker Specificity 7B2 7B2 (γ1) 7B2 (κ) — gp41 CD4-IgG2 CD4-IgG2 CD4 (κ) — gp120 CD4/7B2-Ig CD4-2H2-7B2 CD4-2H2-7B2 2H2* gp41/gp120 #2816 CD4/7B2-Ig CD4-H4-7B2 CD4-H4-7B2 H4* gp41/gp120 #2817 CD4/7B2-Ig 7B2 CD4-2H2-7B2 2H2 gp41/gp120 #2858 CD4/7B2-Ig CD4-2H2-7B2 7B2 2H2 gp41/gp120 #2859 CD4/7B2-Ig 7B2 CD4-H4-7B2 H4 gp41/gp120 #2860 CD4/7B2-Ig CD4-H4-7B2 7B2 H4 gp41/gp120 #2861 *2H2 is two flexible domains flanking a helical core: [(GGGGS)_(x2)-(EAAAK)_(x4)-(GGGGS)_(x2)]., H4 is the helical core only: [A-(EAAAK)_(x4)-A]

Full-length DVD-IgsCD4/7B2 chimeras with appropriately linked subunits were produced in 293F cells in sufficient quantity and purity to test their ability to function as immunoconjugates, bind to infected cells, and neutralize HIV.

Binding of DVD-Igs to Recombinant and Native Antigen

ELISA was used to demonstrate binding of each construct to cognate antigens: gp41 peptide, recombinant gp160, or trimeric gp140. See, e.g., FIG. 1. ELISA plates were coated with respective antigens, incubated with serial dilutions of Abs, and probed with an AP-conjugated anti-human IgG secondary antibody. FIG. 1 has three graphs showing the binding qualities of each construct and parental antibodies to gp41, gp160 and trimeric gp140. All CD4-Ig constructs bound each antigen in the nanomolar range. As expected, CD4-IgG2 does not have any reactivity with gp41, and binds the more “native” trimer gp140 better than non-native gp160. 7B2, which identifies a linear epitope, binds well to the peptide and gp160, but less well to trimeric gp140, suggesting this epitope may be partially occluded in the trimer. There was no preference for antigen binding to gp41 based on attachment of CD4 to the light (#2860) or heavy chain (#2859, 2861), suggesting CD4 does not hinder accessibility of 7B2 to its epitope. Studies with trimeric gp140 showed that CD4/7B2 chimeras with CD4 on the heavy chain only (#2859, 2861) exhibited stronger binding than parental 7B2. And at the highest Ig concentration, #2861 exhibited the strongest binding to all antigens tested. Overall, the DVD-Igs perform as well or better than 7B2 and sCD4-IgG2, especially in the assays using the trimeric form of gp140, suggesting that recognition of the a more native conformation improves cumulative binding.

To test recognition of native Env by the chimeras, indirect immunofluorescence and flow cytometry were used to analyze binding to persistently infected H9/NL4-3 cells. See, e.g., FIG. 2. In addition to the novel constructs, which target both gp41 and gp120, binding of a set of gp120-specific CD4-Ig chimeras designed by others were also examined. It was found that binding of the CD4/Ig chimeras specific solely for gp120 exceeded binding of the constructs specific for both gp120 and gp41. Of the gp120/gp41 specific chimeras, construct #2817, with CD4 fused to both heavy and light chains, bound best.

Immunoconjugate Cytoxicity

To determine whether the novel chimeras with CD4 linked to an anti-Env antibody can function as immunoconjugates to deliver cytotoxic agents to HIV-infected cells as a means to eradicate HIV infection, indirect immunoconjugate assay was used to compare the constructs. H9/NL4-3 cells were incubated with serial dilutions of Ab, then a ricin A chain conjugated to anti-IgG secondary Ab was added. Cell viability was measured after 3 days. 7B2 was highly effective in targeting the toxin when sCD4 was present, but ineffective in its absence. See, e.g., FIG. 3. CD4-Igs exhibited enhanced cytotoxicity compared to either parental antibody alone and to all other CD4-linked constructs. Bispecific CD4/7B2 targeting gp41 and gp120 exhibited more potent cytotoxicity than similar constructs only targeting gp120. No chimera was more effective than 7B2+sCD4. Among the CD4/7B2 constructs, those with two CD4 per construct (#2860 and #2861) outperformed the construct tetravalent with CD4 (#2817). The chimeras to the target cells and immunoconjugate killing did not correlate each other. Those binding to gp120 alone attached to the target cell better than those binding gp120/gp41, but killed the same cells less well. Similarly the CD4/7B2 construct tetravalent for CD4 attached best, but was least affective for delivering toxins.

HIV Neutralization

Although 7B2 has been shown to be an effective immunoconjugate, it cannot effectively neutralize most strains of HIV in conventional assays of neutralization. On the other hand, CD4-IgG2 contains both the gp120 binding site and the immunoglobulin constant region, and makes an effective neutralizing antibody. To test the neutralization ability of CD4/7B2-Ig constructs, both CXCR4 and CCR5-tropic HIV isolates were used with a TZM-b1 luciferase assay. Infectious virus stocks were premixed with dilutions of antibodies, incubated with TZM-b1 cells for 3 days and then luciferase activity was assayed. Constructs with CD4 linked to both chains (#2817), the parental CD4-IgG2, and a panel of other CD4-linked immunoadhesins to four different HIV virus strains were tested first. In all isolates tested, #2817 exhibited excellent neutralization activity and outperformed all other constructs. See, e.g., FIG. 4. In FACS experiments with HIV-infected cells, anti-gp120 constructs showed strong binding. Here they are able to neutralize several isolates at the highest concentrations but quickly lose efficacy. VRC01 is the most effective for Ba-L and HT-594, while CD4/E51 neutralizes QZ4589 and HT-599. However, #2817 consistently displays enhanced neutralization of all four strains across all concentrations. Other configurations of CD4/7B2-Igs were tested against viral isolates ranging in difficulty to neutralize from easy (Ba-L) to hard (HT-92-594). FIG. 5 shows that constructs carrying CD4 on the heavy chain only (#2859 and 2861) neutralized all strains tested more efficiently than #2817 or any other chimeras in the panel, even better than parental CD4-IgG2, which has been used in phase II clinical trials. The most striking results were observed in the most difficult to neutralize strain, HT-92-594. The CD4/7B2-Igs #2859 and #2861 neutralized at >1000× lower concentration than any other CD4 chimeric construct.

Further tests of neutralization were performed at the Duke University HIV neutralization reference laboratories. Parental antibodies (7B2, CD4-IgG2), subunits (sCD4), and CD4/7B2-Igs (#2859, 2860, 2861), were compared to a standard monoclonal antibody mixture (CH01-31). Neutralization of pseudovirus was tested in both TZM-b1 and A3R5.7 cells using tier 1 and 2 viruses from clades B or C. The CD4/7B2-Igs, especially #2861, showed broad and potent neutralizing activity across clades, consistently outperforming the parental antibodies, sCD4, or the Ch01-31 standard. Constructs with CD4 fused to the heavy chain (#2859 and 2861) outperformed the light chain fusion (#2860).

Materials and Methods

Reagents and Cells

H9 cells, human CD4+ lymphoma cell line, were obtained from Dr. M Reitz (Institute of Human Virology, Baltimore, Md.). H9/NL4-3 cells are persistently infected with the NL4-3 molecular clone of HIV and retain a productive infection in virtually 100% of tissue culture cells. TZM-b1 cells (NIH AIDS Reagent Program, NIH-ARP) are HeLa cells expressing CD4, CCR5, and CXCR4, with a HIV-tat inducible luciferase and beta-galactosidase reporter genes. H9/NL4-3 and TZM-b1 cells were maintained at 37° in 5% CO₂ in RPMI 1640 medium with 10% fetal bovine serum (Gibco Invitrogen, Grand Island, N.Y.) as described elsewhere.

HIV isolates used in these studies were all Clade B, and include: NL4-3 (X4-tropic), Ba-L (R5-tropic), 92HT594 (X4/R5), 92HT599 (X4), QZ4589 (R5), and 96USHIPS7 (R5). All isolates were obtained from NIH-ARP and grown in PHA blasts, with the exception of NL4-3, which was produced by the H9/NL4-3 cell line.

Soluble, two-domain CD4 (sCD4; NIH-ARP) and CD4-IgG2 (PRO542; Progenies Pharmaceuticals, Tarrytown, N.Y.) were used to observe CD4-mediated effects. Goat anti-human IgG (heavy+light chains) antibody was conjugated to fluorescein isothiocyanate (FITC; Invitrogen) for flow cytometric analysis. Deglycosylated ricin A chain (RAC; obtained from Ellen Vitetta) was conjugated to purified anti-human IgG for cytotoxicity assays.

Design and Production of Antibodies

Synthetic genes encoding CD4/7B2-Ig constructs shown in table 1 were synthesized, codon optimized for mammalian expression by GenScript (Piscataway, N.J.). Two additional mutations (T250Q and M428L) were introduced into the constant region of the heavy chain to increase in vivo half-life of the antibody. DNA sequences were cloned into the eukaryotic expression plasmid pcDNA3.1 (Invitrogen) using either restriction enzyme sites XbalI and PmeI for the heavy chain, or HindIII and EcoRI for the light chain.

All antibodies were produced by transient transfection in suspension 293F cells (Invitrogen, Carlsbad, Calif.) in serum-free Freestyle expression media, shaking at 120 rpm in 8% Co_(e) at 37′ for transient transfection. Synthetic Igs were purified by affinity chromatography on Protein A agarose beads (Invitrogen), and concentrated by Microcon YM-30k centrifugal filter (Millipore, Billerica, Mass.). All antibody concentrations were measured by bicinchoninic acid protein assay (Pierce, Rockford, Ill.) and confirmed using OD280. Microcapillary electrophoresis (Agilent Bioanalyzer, GE Healthcare) was used to determine molecular weights and purity of products, and confirm concentrations

Domains 1 and 2 of CD4 were joined to 7B2 using two effective linkers from previous studies: SEQ ID NO: 9 or SEQ ID NO: 10. The variable domains of CD4 were fused to the N-terminus of either the 7B2 IgG1 heavy chain, 7B2 kappa light chain, or both, creating a set of full length CD4/7B2-Ig.

ELISA

The antibodies tested, including the novel constructs, were characterized based on binding to Env (gp160) or its subunits (gp41, 140) by indirect ELISA. The gp41 peptide has a linear sequence SEQ ID NO: 3 representing the epitope of 7B2. Gp160 antigen is a recombinant protein consisting of the gp120 portion of MN and the gp41 portion of LAI, designated MN/LAI (Quality Biological, Gaithersburg, Md.) and expressed in mammalian cells. Gp140 is a trimeric version derived from SF162 that was used to test binding to multimers. Immulon 2HB plates (Thermo, Walktham, Mass.) were coated with 1.0 ug/ml of antigen and the assay performed as described elsewhere, using AP-conjugated goat anti-human IgG (H+L chain specific) secondary antibody. ELISA plates were read at 405 nm at room temperature in a BioTek EL320 microplate reader (BioTek, Winooski, Vt.) at 5-15 minute intervals. Time points shown in figures have been chosen so that maximal binding was within the dynamic range of the reader. Data are presented as the mean and SEM of triplicate assays.

Indirect Immunofluorescence and Flow Cytometry

H9/NL4-3 cells (1×10^5) were stained for flow cytometry in 100 ul in round bottom 96 well plates (Costar, Lowell, Mass.). Serial dilutions of Ig in PBA were added to the cells in the presence or absence of 500 ng/ml sCD4. Cells were incubated 1 hr at room temperature, washed, then stained with FITC-conjugated goat anti-human IgG (H+L chain specific) secondary antibody for 1-4 hrs, washed twice and fixed in 100 ul of 2% paraformaldehyde. After a minimum of 4 hrs, 150 ul PBS was added. Cells were analyzed on a Becton-Dickinson LSR II (BD<Franklin Lakes, N.J.) with HTS plate reader. 10000 events were collected and data analyzed by Flo-Jo software (Treestar, Ashland, Oreg.). Forward scatter (FSC) and side scatter (SSC) gated data are represented as graphs of mean fluorescence. None of the parental or synthetic-Igs bound to uninfected H9 cells.

Cytotoxicity Assay

An indirect cytotoxicity assay was performed to screen unconjugated antibodies for their ability to kill infected cells. H9/NL4-3 cells (8×10^3) were plated in triplicate. Controls included: no cells (background) and cells in the absence of antibody/IC (uninhibited). Serial dilutions of antibodies were incubated with cells for 1 hr in the presence or absence of 300 μg/ml sCD4 in RPMI at 37°. The secondary IC was affinity purified goat anti-human IgG (Invitrogen) conjugated to deglycosylated ricin A chain by the long chain heterobifunctional cross linking reagent, succinimidyl 6-[3(2-pyridyldithio)proprionamido]hexanoate (Pierce), using protocols described elsewhere [ ]. The secondary IC was added to a final concentration of 500 ng/ml. The plates were then incubated for 3 days. For the final 6 hrs of incubation, MTS/PMS substrate (Promega, Madison, Wis.) was added to each well and plates read hourly at 490 nm. Results represent the mean and SEM of triplicate samples, and are plotted as “Percent Killing” using the formula % kill=100*[(no Ab−Ab)/No Ab] with the no cell background subtracted. Under these conditions, there was no cytotoxicity on uninfected H9 cells. To determine whether the IC activity of the DVD-Ig represented an improvement over that of the parental antibodies, a one-tailed t-test comparing the DVD-Ig to the most effective parental antibody at each concentration was performed.

Direct cytotoxicity assay was performed with antibodies conjugated to ricin A chain by the long chain heterobifunctional cross linking reagent, succinimidyl 6-[3(2-pyridyldithio)proprionamido]hexanoate (Pierce. H9/NL4-3 cells (8×10^3) were plated in triplicate in cRPMI in 96 well flat-bottom tissue culture plates (Costar). Control included: no cells (background) and cells in the absence of IC, and cells +/−500 μg/ml CD4-IgG2. Serial dilutions of ICs were incubated with cells for 3 days in RPMI at 37°. For the final 6 hrs of incubation, MTS/PMS substrate (Promega) was added to each well and plates read hourly at 490 nm. Results plotted similarly to indirect IC assay explained above.

Neutralization Assay

Neutralization of infectious HIV was measured in TZM-b1 cells, using a luciferase-read out assay. Each antibody was assayed in triplicate. Experiments included: background controls (cells, no virus, no antibody) and infected cells in the presence or absence of antibody. TZM-b1 cells (4×10^4 cells/ml) were plated in 96-well plates with black sides and clear, flat bottom wells (Costar) and incubated overnight at 37° to allow attachment. The following day, 50 μl of serially diluted antibodies in RPMI were mixed with 50 μl of a pretitered concentration of virus and incubated for 1 hr at room temperature, then added to the cells in the presence of diethylaminoethyl dextran (Sigma) 15 μg/ml, and incubated for 6 hrs at 37°. Medium was added to a total volume of 200 ul/well and plates incubated for 48 hr at 37°. For luciferase assays, medium was aspirated and 50 μl of Bright-Glo Lysis buffer (Promega) was added. Samples were frozen and thawed once, and incubated for 6 hr at room temperature with orbital shaking at 120 rpm. Then 10 ul of Bright-Glo luciferase substrate (Promega) was added and luminescence read on Bio-Tek KC4 plate reader as relative luminescence units. Results are displayed as percent neutralization (virus/no Ab=0%; no virus=100% neutralization) according to the formula: [1−(RLUAb−RLUbkgrd)/(RLUnoAb−RLUbackground)]*100.

Neutralization of pseudo-typed reference strains in TZM-b1 and A3R5.7 cells was performed at the Duke University HIV neutralization reference laboratories, using established assays.

Antibody-Dependent Cellular Viral Inhibition (ADCVI)

Embodiments of the present invention were tested to determine their ability to mediate ADCVI using human peripheral blood mononuclear cells (PBMC) as effector cells and CCR5+ CEM-NKr cells (AIDS Research and Reference Reagent Program) infected 2 days earlier with HIVBAL at an moi of 0.02 as target cells. See, e.g., FIG. 6. Briefly, target cells were washed twice in medium, then placed in wells of a V bottom plate at 104 cells per well. Target cells were incubated in triplicate with medium alone or diluted antibodies for 1 h at 37° C. and 5% CO₂. Freshly isolated PBMC (105 per well) were then added. Four days later, the cultures were split ¼. On day 7, the medium in the wells was harvested, lysed with TritonX-100 detergent and analyzed for p24 content by ELISA. The % inhibition of infection was calculated after dividing the p24 concentration in antibody cultures by the average p24 concentration in control cultures containing effector and target cells alone. Construct no. 2861 showed greater efficacy than either of the parental antibodies, or a mixture of both. See, e.g., FIG. 6.

Antibody-Dependent Phagocytosis (ADP)

Assays were performed using the THP-1 monocyte cell line and HIV gp140-coated fluorescent beads to evaluate ADP, specifically for construct no. 2861. See, e.g., FIG. 7. Briefly, 1.8×106 neutravidin-coated 1 μm Fluorospheres (Invitrogen) were treated with rabbit anti-His tag antibody (Pierce), then washed and reacted with recombinant gp140 SF162 protein (Immune Technology). After washing unbound material off the beads, they were incubated at 37° C. for 1 h with dilutions of antibody in triplicate wells of a V-bottom plate. THP-1 cells (2×104 per well) were then added and incubated at 37° C. in 5% CO2. After 4 h, the cells were washed with Ca+2/Mg+2-free DPBS and incubated at 37° C. for 10 min with 50 μl of 0.05% Trypsin/EDTA (Life Technologies). Cells were washed 2× in DPBS, re-suspended in 1% paraformaldehyde, then examined by flow cytometry for fluorescence. The phagocytic score was calculated by multiplying the number of bead-positive cells by the median fluorescent intensity. The average score obtained for triplicate wells of THP-1 and gp140-coated beads incubated in medium alone was subtracted from all other scores prior to calculating the average score for test samples. The ability of construct no. 2861 to promote phagocytosis of HIV-Env is greater than that of the combination of the two parental Abs. See, e.g., FIG. 7.

While the embodiments are described with reference to various implementations and exploitations, it will be understood that these embodiments are illustrative and that the scope of the invention is not limited to them. Many variations, modifications, additions, and improvements are also possible. Support for the present invention may be found in the attached documents and figures, all of which are expressly incorporated herein in their entirety by reference hereto.

Sequence Listing SEQ ID NO 1 Length: 345 Type: amino acid Organism: Human Immunodeficiency Virus Other Information: gp 41 of Env         10         20         30         40         50         60         70          |          |          |          |          |          |          |   AVGIGALFLG FLGAAGSTMG AASMTLTVQA RQLLSGIVQQ QNNLLRAIEA QQHLLQLTVW GIKQLQARIL         80         90        100        110        120        130        140          |          |          |          |          |          |          |   AVERYLKDQQ LLGIWGCSGK LICTTAVPWN ASWSNKSLEQ IWNHTTWMEW DREINNYTSL IHSLIEESQN        150         160       170        180        190        200        210          |          |          |          |          |          |          |   QQEKNEQELL ELDKWASLWN WFNITNWLWY IKLFIMIVGG LVGLRIVFAV LSIVNRVRQG YSPLSFQTHL        220        230        240        250        260        270        280          |          |          |          |          |          |          |   PTPRGPDRPE GIEEEGGERD RDRSIRLVNG SLALIWDDLR SLCLFSYHRL RDLLLIVTRI VELLGRRGWE        290        300        310        320        330        340          |          |          |          |          |          | ALKYWWNLLQ YWSQELKNSA VSLLNATAIA VAEGTDRVIE VVQGACRAIR HIPRRIRQGL ERILL SEQ ID NO 2 Length: 481 Type: amino acid Organism: Human Immunodeficiency Virus Other Information: gp 120 of Env         10         20         30         40         50         60         70          |          |          |          |          |          |          |   TEKLWVTVYY GVPVWKEATT TLFCASDAKA YDTEVHNVWA THACVPTDPN PQEVVLVNVT ENFNMWKNDM         80         90        100        110        120        130        140          |          |          |          |          |          |          |   VEQMHEDIIS LWDQSLKPCV KLTPLCVSLK CTDLKNDTNT NSSSGRMIME KGEIKNCSFN ISTSIRGKVQ        150         160       170        180        190        200        210          |          |          |          |          |          |          |   KEYAFFYKLD IIPIDNDTTS YKLTSCNTSV ITQACPKVSF EPIPIHYCAP AGFAILKCNN KTFNGTGPCT        220        230        240        250        260        270        280          |          |          |          |          |          |          |   NVSTVQCTHG IRPVVSTQLL LNGSLAEEEV VIRSVNFTDN AKTIIVQLNT SVEINCTRPN NNTRKRIRIQ        290        300        310        320        330        340        350          |          |          |          |          |          |          | RGPGRAFVTI GKIGNMRQAH CNISRAKWNN TLKQIASKLR EQFGNNKTII FKQSSGGDPE IVTHSFNCGG        360        370        380        390        400        410        420          |          |          |          |          |          |          | EFFYCNSTQL FNSTWFNSTW STEGSNNTEG SDTITLPCRI KQIINMWQKV GKAMYAPPIS GQIRCSSNIT        430        440        450        460        470        480          |          |          |          |          |          | GLLLTRDGGN SNNESEIFRP GGGDMRDNWR SELYKYKVVK IEPLGVAPTK AKRRVVQREK SEQ ID NO 3 Length: 710 Type: amino acid Other Information: CD4-[2-helix-2]-7B2 DVR Heavy Chain         10         20         30         40         50         60         70          |          |          |          |          |          |          | MNRGVPFRHL LLVLQLALLP AATQGKKVVL GKKGDTVELT CTASQKKSIQ FHWKNSNQIK ILGNQGSFLT         80         90        100        110        120        130        140          |          |          |          |          |          |          | KGPSKLNDRA DSRRSLWDQG NFPLIIKNLK IEDSDTYICE VEDQKEEVQL LVFGLTANSD THLLQGQSLT        150         160       170        180        190        200        210          |          |          |          |          |          |          | LTLESPPGSS PSVQCRSPRG KNIQGGKTLS VSQLELQDSG TWTCTVLQNQ KKVEFKIDIV VLAFQKASGG        220        230        240        250        260        270        280          |          |          |          |          |          |          | GGSGGGGSLE AEAAAKEAAA KEAAAKEAAA KALEGGGGSG GGGSQVQLVQ SGGGVFKPGG SLRLSCEASG        290        300        310        320        330        340        350          |          |          |          |          |          |          | FTFTEYYMTW VRQAPGKGLE WLAYISKNGE YSKYSPSSNG RFTISRDANK NSVFLQLDRL SADDTAVYYC        360        370        380        390        400        410        420          |          |          |          |          |          |          | ARADGLTYFS ELLQYIFDLW GQGARVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS        430        440        450        460        470        480        490          |          |          |          |          |          |          | WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP        500        510        520        530        540        550        560          |          |          |          |          |          |          | PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN        570        580        590        600        610        620        630          |          |          |          |          |          |          | STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC        640        650        660        670        680        690        700          |          |          |          |          |          |          | LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT        710          | QKSLSLSPGK SEQ ID NO 4 Length: 690 Type: amino acid Other Information: CD4-DVR Heavy Chain         10         20         30         40         50         60         70          |          |          |          |          |          |          |   MNRGVPFRHL LLVLQLALLP AATQGKKVVL GKKGDTVELT CTASQKKSIQ FHWKNSNQIK ILGNQGSFLT         80         90        100        110        120        130        140          |          |          |          |          |          |          |   KGPSKLNDRA DSRRSLWDQG NFPLIIKNLK IEDSDTYICE VEDQKEEVQL LVFGLTANSD THLLQGQSLT        150         160       170        180        190        200        210          |          |          |          |          |          |          |   LTLESPPGSS PSVQCRSPRG KNIQGGKTLS VSQLELQDSG TWTCTVLQNQ KKVEFKIDIV VLAFQKASLE        220        230        240        250        260        270        280          |          |          |          |          |          |          |   AEAAAKEAAA KEAAAKEAAA KALEQVQLVQ SGGGVFKPGG SLRLSCEASG FTFTEYYMTW VRQAPGKGLE        290        300        310        320        330        340        350          |          |          |          |          |          |          |   WLAYISKNGE YSKYSPSSNG RFTISRDNAK NSVFLQLDRL SADDTAVYYC ARADGLTYFS ELLQYIFDLW        360        370        380        390        400        410        420          |          |          |          |          |          |          |   GQGARVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS        430        440        450        460        470        480        490          |          |          |          |          |          |          |   GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP        500        510        520        530        540        550        560          |          |          |          |          |          |          |   KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK        570        580        590        600        610        620        630          |          |          |          |          |          |          |   EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP        640        650        660        670        680        690                 |          |          |          |          |          |          ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK SEQ ID NO 5 Length: 502 Type: amino acid Other Information: CD4-[2-helix-2]-7B2 DVR Light Chain         10         20         30         40         50         60         70          |          |          |          |          |          |          | MTSTLPFSPQ VSTPRSKFKR ISSEFAATMN RGVPFRHLLL VLQLALLPAA TQGKKVVLGK KGDTVELTCT         80         90        100        110        120        130        140          |          |          |          |          |          |          | ASQKKSIQFH WKNSNQIKIL GNQGSFLTKG PSKLNDRADS RRSLWDQGNF PLIIKNLKIE DSDTYICEVE        150         160       170        180        190        200        210          |          |          |          |          |          |          | DQKEEVQLLV FGLTANSDTH LLQGQSLTLT LESPPGSSPS VQCRSPRGKN IQGGKTLSVS QLELQDSGTW        220        230        240        250        260        270        280          |          |          |          |          |          |          | TCTVLQNQKK VEFKIDIVVL AFQKASGGGG SGGGGSLEAE AAAKEAAAKE AAAKEAAAKA LEGGGGSGGG        290        300        310        320        330        340        350          |          |          |          |          |          |          | GSDIVMTQSP DSLAVSPGER ATIHCKSSQT LLYSSNNRHS IAWYQQRPGQ PPKLLLYWAS MRLSGVPDRF        360        370        380        390        400        410        420          |          |          |          |          |          |          | SGSGSGTDFT LTINNLQAED VAIYYCHQYS SHPPTFGHGT RVELRRTVAA PSVFIFPPSD EQLKSGTASV         430        440        450        460        470        480        490          |          |          |          |          |          |          | VCLLNNFYPR EAKVQWKVDN ALQSGNSQES VTEQDSKDST YSLSSTLTLS KADYEKHKVY ACEVTHQGLS        500          | SPVTKSFNRG EC SEQ ID NO 6 Length: 482 Type: amino acid Other Information: CD4-DVR Light Chain         10         20         30         40         50         60         70          |          |          |          |          |          |          |   MTSTLPFSPQ VSTPRSKFKR ISSEFAATMN RGVPFRHLLL VLQLALLPAA TQGKKVVLGK KGDTVELTCT         80         90        100        110        120        130        140          |          |          |          |          |          |          |   ASQKKSIQFH WKNSNQIKIL GNQGSFLTKG PSKLNDRADS RRSLWDQGNF PLIIKNLKIE DSDTYICEVE        150         160       170        180        190        200        210          |          |          |          |          |          |          |   DQKEEVQLLV FGLTANSDTH LLQGQSLTLT LESPPGSSPS VQCRSPRGKN IQGGKTLSVS QLELQDSGTW        220        230        240        250        260        270        280          |          |          |          |          |          |          |   TCTVLQNQKK VEFKIDIVVL AFQKASLEAE AAAKEAAAKE AAAKEAAAKA LEDIVMTQSP DSLAVSPGER        290        300        310        320        330        340        350          |          |          |          |          |          |          |   ATIHCKSSQT LLYSSNNRHS IAWYQQRPGQ PPKLLLYWAS MRLSGVPDRF SGSGSGTDFT LTINNLQAED        360        370        380        390        400        410        420          |          |          |          |          |          |          |   VAIYYCHOYS SHPPTFGHGT RVELRRTVAA PSVFIFPPSD EQLKSGTASV VCLLNNFYPR EAKVQWKVDN        430        440        450        460        470        480                  |          |          |          |          |          |          ALQSGNSQES VTEQDSKDST YSLSSTLTLS KADYEKHKVY ACEVTHQGLS SPVTKSFNRG EC SEQ ID NO 7 Length: 240 Type: nucleic acid Other Information: 7B2 Light Chain         10         20         30         40         50         60         70          |          |          |          |          |          |          |   METPAQLLFL LLLWLPDTTG DIVMTQSPDS LAVSPGERAT IHCKSSQTLL YSSNNRHSIA WYQQRPGQPP         80         90        100        110        120        130        140          |          |          |          |          |          |          |   KLLLYWASMR LSGVPDRFSG SGSGTDFTLT INNLQAEDVA IYYCHQYSSH PPTFGHGTRV ELRRTVAAPS        150         160       170        180        190        200        210          |          |          |          |          |          |          |   VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA        220        230        240          |          |          | DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC SEQ ID NO 8 Length: 475 Type: amino acid Other Information: Parent 7B2 Heavy Chain         10         20         30         40         50         60         70          |          |          |          |          |          |          | MDWTWRVLFL VAAATGAHSQ VQLVQSGGGV FKPGGSLRLS CEASGFTFTE YYMTWVRQAP GKGLEWLAYI         80         90        100        110        120        130        140          |          |          |          |          |          |          | SKNGEYSKYS PSSNGRFTIS RDNAKNSVFL QLDRLSADDT AVYYCARADG LTYFSELLQY IFDLWGQGAR        150         160       170        180        190        200        210          |          |          |          |          |          |          | VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL        220        230        240        250        260        270        280          |          |          |          |          |          |          | SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KRVEPKSCDK THTCPPCPAP ELLGGPSVFL FPPKPKDTLM        290        300        310        320        330        340        350          |          |          |          |          |          |          | ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK         360        370        380        390        400        410        420          |          |          |          |          |          |          | VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK        430        440        450        460        470                       |          |          |          |          |              TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK SEQ ID NO 9 Length: 22 Type: amino acid Other Information: helical linker         10         20          |          | LEAEAAAKEA AAKEAAAKEA AAKALE SEQ ID NO 10 Length: 46 Type: amino acid Other Information: 2-Helix-2 linker         10         20          30         40          |          |           |          | GGGGSGGGGS LEAEAAAKEA AAKEAAAKEAI AAKALEGGGG SGGGGS 

What is claimed is:
 1. An asymmetric double variable domain (DVD) antibody comprising at least one immunoglobulin heavy chain-light chain cross-linked pair, wherein each immunoglobulin heavy chain-light chain pair comprises: a first variable domain having binding affinity for an epitope of an HIV gp41 polypeptide and consisting of an immunoglobulin heavy chain variable (V_(H)) region and an immunoglobulin light chain variable (V_(L)) region; and a second variable domain having binding affinity for an epitope of an HIV gp120 polypeptide, wherein said second variable domain is attached by a linker peptide to either the immunoglobulin V_(H) region or the immunoglobulin V_(L) region of the first domain, but is not attached to both the immunoglobulin V_(H) region and the immunoglobulin V_(L) region of the first variable domain.
 2. The asymmetric DVD-antibody of claim 1, wherein the second variable domain having binding affinity for an epitope of an HIV gp120 polypeptide is a CD4 polypeptide or a fragment thereof.
 3. The asymmetric DVD-antibody of claim 1, wherein the first variable domain having binding affinity for an epitope of an HIV gp41 polypeptide is the immunoglobulin variable region of monoclonal antibody 7B2.
 4. The asymmetric DVD-antibody of claim 1, wherein the V_(H) region of the first variable domain has the second variable domain attached thereto by the linker peptide and the the V_(L) region of the first variable domain does not have the second variable domain attached thereto.
 5. The asymmetric DVD-antibody of claim 1, wherein the V_(L) region of the first variable domain has the second variable domain attached thereto by the linker peptide, and the the V_(H) region of the first variable domain does not have the second variable domain attached thereto.
 6. The asymmetric DVD-antibody of claim 1, wherein the linker peptide comprises a helical core.
 7. The asymmetric DVD-antibody of claim 1, wherein the linker peptide comprises a helical core having a flexible domain at both the N-terminus and the C-terminus thereof.
 8. The asymmetric DVD-antibody of claim 1, wherein the heavy chain has an amino acid sequence selected from the group consisting of: SEQ ID Nos.: 3, 4, and 8, and wherein the light chain has an amino acid sequence selected from the group consisting of: SEQ ID Nos.: 5, 6, and
 7. 9. The asymmetric DVD-antibody of claim 1, wherein the cross-linked heavy and light chain pairs are paired amino acid sequences-selected from the group consisting of: SEQ ID Nos.: 3 and 5, 4 and 6, 8 and 5, 3 and 7, 8 and 6, and 4 and
 7. 10. The asymmetric DVD-antibody of claim 9, wherein the cross-linked heavy and light chain pairs are paired amino acid sequences selected from the group consisting of: SEQ ID Nos.: 8 and 5, 3 and 7, 8 and 6, and 4 and
 7. 11. The asymmetric DVD-antibody of claim 1, wherein the linker peptide has an amino acid sequence according to SEQ ID NO:
 9. 12. The asymmetric DVD-antibody of claim 1, wherein the linker peptide has an amino acid sequence according to SEQ ID NO:
 10. 13. The asymmetric DVD-antibody of claim 1, wherein the asymmetric immunoglobulin is admixed with a pharmaceutically acceptable carrier.
 14. The asymmetric DVD-antibody of claim 1, wherein each of the heavy and the light chains is an expression product from an expression vector.
 15. The asymmetric DVD-antibody of claim 14, wherein at least one of the heavy and the light chain expression products from the expression vector has an N-terminal leader sequence attached thereto.
 16. A method of reducing the infectivity of an HIV by contacting said virus with an asymmetric DVD-antibody comprising at least one immunoglobulin heavy chain-light chain cross-linked pair, wherein each immunoglobulin heavy chain-light chain pair comprises: a first variable domain having binding affinity for an epitope of an HIV gp41 polypeptide and consisting of an immunoglobulin heavy chain variable (V_(H)) region and an immunoglobulin light chain variable (V_(L)) region; and a second variable domain having binding affinity for an epitope of an HIV gp120 polypeptide, wherein said second variable domain is attached by a linker peptide to either the immunoglobulin V_(H) region or the immunoglobulin V_(L) region of the first domain, but is not attached to both the immunoglobulin V_(H) region and the immunoglobulin V_(L) region of the first variable domain.
 17. A method of reducing the viability of an HIV-infected cell by the steps of: binding an asymmetric DVD-antibody to a cell infected with a strain of HIV, wherein the asymmetric DVD-antibody comprises at least one immunoglobulin heavy chain-light chain cross-linked pair, wherein each immunoglobulin heavy chain-light chain pair comprises: a first variable domain having binding affinity for an epitope of an HIV gp41 polypeptide and consisting of an immunoglobulin heavy chain variable (V_(H)) region and an immunoglobulin light chain variable (V_(L)) region; and a second variable domain having binding affinity for an epitope of an HIV gp120 polypeptide, wherein said second variable domain is attached by a linker peptide to either the immunoglobulin V_(H) region or the immunoglobulin V_(L) region of the first domain, but is not attached to both the immunoglobulin V_(H) region and the immunoglobulin V_(L) region of the first variable domain, whereby said DVD-antibody binds to a cell surface polypeptide of the infected cell forming a cell-DVD-antibody complex; and delivering a cytotoxic agent to the HIV-infected cell and reducing the viability of the HIV-infected cell by contacting said cell-DVD-antibody complex with a cytoxic agent capable of specifically recognizing the cell-bound DVD-antibody.
 18. The method of claim 17, wherein the cytotoxic agent is an engineered anti-immunoglobulin IgG antibody conjugated to a cytotoxic polypeptide, wherein the engineered anti-immunoglobulin IgG antibody specifically binds to a constant region of the asymmetric DVD-antibody.
 19. The method of claim 18, wherein the cytotoxic polypeptide is a ricin A chain. 